Transcript C14, C14:1

Newborn Screening
Historical, Ethical, Technological Aspects
Nutrition 526
November 9, 2009
Cristine M Trahms, MS, RD
Beth Ogata, MS, RD
Lisa Sniderman King, CGC
First screen must
be taken 24-48
hours of life
regardless of
feeding status or
weight
Blood Sample on
Guthrie
Filter Paper Card
Unsatisfactory Specimens
(Provided by the New York State Department of Health)
Supersaturated
Quantity Insufficient for Testing
Specimen Not Dried Before Mailing
Scratched or Abraded
Serum Rings
Diluted, Discolored, or Contaminated
Clotted or Layered
No Blood
Criteria for Newborn Screening
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important condition
acceptable treatment available
facilities for diagnosis and treatment
difficult to recognize early
suitable screening test
natural history known
cost effective to diagnose and treat
Wilson & Jungner, 1968
MS/MS -High Impact and High
Throughput
 One disease, one test is not cost
effective
 Many diseases, one test is cost
effective
 MS/MS allows for rapid, simultaneous
analysis and detection of many
disorders of amino acid, organic acid,
and fatty acid metabolism.
What is MS/MS ?
3/16” blood spot
deproteinization
derivatization
CH3
CH3
R - COO
+N
- CH2 - CH - CH2 - COO - C4H9
CH3
CH3
CH3
R - COO
+N
- CH2 - CH - CH2 - COO - C4H9
Data of all compounds
within selected range
(250 - 500 m/z)
- CH - CH2 - COOH
(m/z 85)
CH3
Mass Spectrometer 1
+CH
2
Collision Cell

Mass Spectrometer 2
Data of product ions
with a mass of 85 only
Data system correlates m/z 85 to its precursor ion’s mass and records the
abundance of all precursors (parents of m/z 85)
Tandem Mass Spectrometry
(MS/MS)
 Compounds analyzed are amino
acids & acylcarnitines
 Amino acids – PKU, MSUD,
Homocystinuria
 Acylcarnitine {Carnitine (vehicle)
+fatty acid} for identification of
organic acidurias and fatty acid
oxidation disorders.
Amino Acid Disorders
 AA that are not used to make proteins are recycled by
their specific metabolic pathways. Enzymatic
deficiencies in these pathways lead to various clinical
phenotypes.
 PKU – Phenylketonuria : severe perm MR
 MSUD – Maple syrup urine disease: dd, hallucinations,
ataxia
 HCY – Homocystinuria: connective tissue damage –
joints, heart, dd, psychiatric dist.
 CIT – Citrullinemia: risk of hyperammonemiadd, coma,
death
 ASA – Argininosuccinic acidemia: brittle hair, liver dis, dd
 TYR I – Tyrosinemia type I: acute or chronic liver disease,
liver cancer, neurologic pain crises.
 Diagnosed by plasma amino acids, and/or urine amino
acids, and/or urine organic acids (takes 2-5 days)
Organic Acid Disorders
 Organic acids are breakdown products of protein and
fatty acid metabolism. Defects in their breakdown lead
to (generally)
 Vomiting, metabolic acidosis, elevated ammonia in
crises
 Dd, motor delay, ataxia, heart/kidney/pancreatic
problems
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IVA - Isovaleric acidemia
GA I – Glutaric acidemia type I
HMG – 3-OH 3-CH3 glutaric aciduria
MCD – Multiple carboxylase deficiency
MUT – Methylmalonic acidemia (mutase def)
3MCC – 3-Methylcrotonyl-CoA carboxylase deficiency
Cbl A,B – Methylmalonic acidemia
PROP – Propionic acidemia
BKT – Beta-ketothiolase deficiency
 Diagnosed by urine organic acids and/or plasma
Fatty Acid Oxidation
Disorders
 Fatty acid disorders lead to impaired energy production.
 Hypoglycemia, cardiomyopathy, muscle weakness can
be seen
 MCAD – Medium-chain acyl-CoA dehydrogenase
deficiency
 VLCAD – Very long-chain acyl-CoA dehydrogenase
deficiency
 LCHAD – Long-chain L-3-OH acyl-CoA dehydrogenase
deficiency
 TFP – Trifunctional protein deficiency
 CUD – Carnitine uptake defect
 Diagnosed by plasma acylcarnitines and urine organic
acids can be helpful.
MS/MS Plasma Acylcarnitines
100%
C8
Intensity
*
C2
MCAD
* *
C6
*
C10:1
*
*
C16
100%
Control
Intensity
*
C2
* *
*
*
*
* internal standards
C16
*
Normal
VLCAD profile
Free
Carnitine
C2
*
C3
*
C4
*
C8
*
C12
*
*
VLCAD
C12 C14:1C16 C18:1
Free
Carnitine
*
*
*
*
*
* internal standards
MS/MS Plasma Amino Acids
Acylcarnitine – VLCAD Deficiency
Which Disorders to Screen For?
 NBS mandates are under state
control
 Some states screened for 3 diseases,
others 40+
 2002 Maternal and Child Health
Bureau commissioned ACMG
 Analyze literature
 Develop consensus on which disorders
 Recommend a core panel to create
uniform NBS across all states.
Historical Harm (?)
 Early PKU screening led to cases of
overly restricted phe and/or
implementation of diet prior to
confirmation of diagnosis
 Today, diagnosis is quite rapid
 40 years ago it took much longer so more
potential for harm
 However, no published evidence of
wide-spread physical/medical harm
 BUT the cases do underscore need for
expertise and resources for mgmt
Whom do we see?
1. Patients who need active
management
 Symptomatic at diagnosis
 Strong evidence of pathology if
untreated
 Examples: PKU, classic gal, MSUD, PA
etc.
Whom do we see?
2. Patients with disorders known to
pose risk but reduced penetrance
 ie. probably not everyone needs to be
treated
 HPHE, MCAD
 Both are/have mild ends of the
spectrum that have only been
identified through NBS
 MCAD mutation c.199 C>T
 Never seen in patients picked up
clinically
Whom do we see?
3. Patients who may not need any
management
 Disorders considered extremely rare
but seen in large numbers via NBS
programs
 Reported cases have significant
morbidity
 NBS pickups are mostly mild
 3MCC, SCAD
 Biochemical phenotype
Proceeding with Caution
(Reasons to be Thoughtful)
Proceeding with caution  Not screening
 Core diseases vs secondary
targets/unintended targets
 What is reported vs withheld?
 Will we p/u untreatable conditions?
 What is the impact of false positives on
families?
 No long-term outcome data – consider
research paradigm
 Consider infrastructure needed for f/u
Other Benefits to Screening
For disorders in which proven, effective
treatment is not available, or very
new.
 Consider non-medical benefits:
 Avoid the diagnostic odyssey
 Allow for reproductive decision making
before future children are born
 Allow for early access to clinical trials
for new therapies
 Emotional preparation for disease
What Are We Screening For?
9 OA
5 FAO
6 AA
3 Hb
Pathies
6
Others
CORE PANEL
IVA
GA I
HMG
MCD
MUT
3MCC
Cbl A,B
PROP
BKT
MCAD
VLCAD
LCHAD
TFP
CUD
PKU
MSUD
HCY
CIT
ASA
TYR I
Hb SS
Hb S/ßTh
Hb S/C
CH
BIOT
CAH
GALT
HEAR
CF
What Are We Measuring?
Disorder
1° Marker
2° Marker
Ratios
VLCADD
C14:1
C14,C16,C18,C18:1
C14:1/C16
LCHADD
C16OH
C14,C14:1,C16:C18,C18:1,C18:1OH, C18OH
C16OH:C16
TFP
C16OH
C14,C14:1,C16:C18,C18:1,C18:1OH, C18OH
C16OH:C16
CUD
C0
All carnitines (low)
PA
C3
C2
C3/C0, C3/C2, C3/C16
MMAs
C3
C4DC
C3/C0, C3/C2, C3/C16
IVA
C5
C5/C2, C5/C0, C5/C3
GA-1
C5DC
C5DC/C8, C5DC/C16, C5DC/C5OH
BKT
C5:1
C5OH
C5OH/C8
HMG
C5OH
C6DC
C5OH/C8
HCSD
C5OH
C3
C5OH/C8
CIT
Cit
ASA
Cit
Asa
TYR
Suac
Tyr
Cit/arg
Cit/arg, cit/phe, cit/tyr, asa/arg
Emma
13 months old
Normal pregnancy and delivery
Healthy
Normal eating pattern, no allergies or intolerances
Feb 2008: Vomited Saturday and 4-5 times
throughout the weekend
 No fever
 Sleeping for extended periods – parents concerned
but previous fever had same pattern.
 Parents gave Pedialyte
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Emma
 4 ½ y brother, parents sick on
Sunday/Monday. Same symptoms
 Monday night 9:30 checked on E
 Raspy breathing – thought respiratory
problem but not worried
 Tuesday morning 11am she was
found motionless in her crib and
pronounced dead at the scene
Emma
 Autopsy revealed fatty changes to
liver
 Coroner requested newborn screening
blood spot be sent for acylcarnitine
profile
 Diagnostic for Very Long Chain AcylCo A Dehydrogenase Deficiency
(VLCAD)
VLCAD
 Disorder of long chain fatty acid
breakdown
 C14, C14:1 C16, C18
 Normal beta oxidation occurs in
mitochondria
Fatty Acid Oxidation
 During times of fasting, fatty acids
are primary substrate for energy
production in liver, cardiac muscle
and skeletal muscle
 Brain uses ketones (produced by
normal b-oxidation)
Fatty Acid Oxidation
http://www.genomeknowledge.org/figures/saturatedbetao.jpg
VLCAD Enzyme
 VLCAD enzyme sits on inner mitochondrial
membrane
 Catalyzes first step of b-oxidation for C14C20
 Defect leads to
 impaired energy production during times of
fasting stress
 Accumulation of toxic long-chain acyl-CoA
intermediates within mitochondria
 Steatosis (fatty accumulation/degeneration)
seen in hepatic, cardiac and skeletal muscle
VLCAD Presentations
 Hypertrophic cardiomyopathy, with
hypoglycemia and skeletal myopathy,
lethargy, failure to thrive
 Usually present birth-5 months
 Hypoglycemia, hepatomegaly, muscle
weakness without cardiac manifestations
 Late infancy – older childhood
 Muscle weakness/pain, rhabdomyolysis with
exercise or illness. No hypoglycemia or
cardiac
 Teens to adulthood
VLCAD Treatment
 Diet low in long-chain fats (Portagen,
Monogen = 87%, 90% of fats as
MCT)
 Additional medium chain fats (MCT
oil, walnut oil)
 Carnitine 100 mg/kg/day
 Avoidance of fasting
 Treating illness with IV glucose
support
VLCAD Diagnosis
 Newborn screening
 Plasma acylcarnitine profile
 Urine organic acids (should be
normal)
 DNA sequencing
Zach Testing
 Family referred to genetics by coroner
 Parents requested testing for older
brother
 Acylcarnitine ordered
 DNA sequencing of ACADVL gene
ordered
Normal acylcarnitine profile
Acylcarnitine – Zach 5 y.o.
C14:1 
C14 
C16 - nl
C16:1- nl
Zach Testing
 Reported: mild elevation of C14 and
C14:1 with low free carnitine. VLCAD
cannot be ruled out
 Recommend supplementing with
carnitine and retest in 1 week
 Family left for Disneyland
 DNA testing results back before AC
repeat
Zach Testing
 Zach’s DNA testing reveal he is
affected.
 Family seen in BCG clinic, started on
treatment.
 Consent to obtain NBS blood spot
obtained
Acylcarnitine – Zach 5 y.o.
C14:1 
C14 
C16 - nl
C16:1- nl
C18 - nl
Acylcarnitine – Zach newborn
C16 
C14:1 
C14 
C18 
C16:1 
Zach Clinical picture
 5 y.o
 Healthy
 No symptoms of muscle weakness
 CPK = 315U/L (35-230)
 No hepatomegaly
 AST= 49 (5-41)
 ALT= 23
 Bilirubin conj, unconj = normal (0.0, 0.4)
 No evidence of cardiac involvement
 Has had several viral illnesses in his lifetime without
difficulty
 Once on carnitine, AC profile was classic for VLCAD
Newborn Screening – A Team
Effort
1. The Presumptive Positive Phase
DOH NBS Laboratory Personnel
Mike Glass, Sheila Weiss, John Thompson, CarolNucup-Villaruz, Charlene Adams, Jessica Dolle,
many laboratory technologists
2. The Diagnostic Confirmation Phase
CHRMC Diagnostic Laboratory Personnel
Sihoun Hahn, Rhona Jack, Lisa Sniderman King, Cindy
Gordon, Nancy McDowell Laura Mitchell, Diane
Rebholz, Malcolm Reider
Monica Jensen, Ngoc-Diep Pham, Min Zhang
Newborn Screening – A Team
Effort
3.
The Clinical Follow-up Phase
Clinic Personnel
All previously screened disorders: (PKU, MCAD,
gal, btd, msud, hcys):
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UW: Ron Scott, Cris Trahms, Beth Ogata, Janie
Heffernan, Jan Garretson, Stefanie Uhrich, Angie
Fox
All expanded screening disorders (FAOs, OAs…etc):
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CHRMC: Lawrence Merritt, Michael Raff, Sihoun
Hahn, Sue Hale, Kelly McKean, Melissa Edwards,
Lisa Sniderman King, Penny Schubert
What Happens After a Positive
1. NBS Lab notifies all clinical f/u and
key laboratory personnel of referral.
2. Laboratory technologists prioritize
samples and collate results
 Multiple tests (AA, OA, AC) on each
kiddo
 Interpreted together once all are
completed
 Uniform, concise reporting
2008
Cases
 58 total cases since Jan
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51 since expanded NBS started (July 21)
8 true positives for targeted disorders (MCAD and PKU)
1 true positive for secondary disorder (CblC)
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Elev C3 1° targets MMA/PA/CblA,B
38 FP/FPA – targeted disorder ruled out
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False Positive Active
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Persistent elevations in ‘normal’ baby
Carriers (ie. further testing needed)
Benign forms (D/G galactosemia)
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False Positive
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These are active because they require genetic counseling or lab
repeats
They are reclassified as FP when case is closed
1 mom with low free carnitine
Several D/G galactosemia
A few VLCAD carriers
10 pending (waiting for samples)
2 Unknown
2008 Cases
Cases
Newborn Screening Cases per Month
24
22
20
18
16
14
12
10
8
6
4
2
0
Jan
Feb
Mar
Apr
May
Jun
Jul
Month
True Positive
Pending
False Positive
Unknown
Aug
Sep
Oct
Nov
Dec